Covalent modification by isoprenoid lipids, a process termed prenylation, is an important component in the localization and activity of many proteins involved in signal transduction pathways. The discovery that the members of the Ras family of oncogene products were modified by the farnesyl (15-carbon) prenyl group, and that prenylation was required for the Ras protein to express their transforming potential, has revealed a previously unrecognized mechanism for regulation of cell growth. Furthermore, a number of other proteins that play critical roles in cellular signaling processes have also been found to be subject to this processing, and a variety of studies have highlighted the importance of the attached isoprenoid in the function of these proteins. These findings have in turn led to the targeting of the enzymes responsible for these modifications in development of therapeutics against a variety of pathologies, mostly notably cancer. The long term goal of this research project is to elucidate the molecular mechanisms of protein prenylation. The primary routes to achieve this goal initially involved identification and cloning of the two main protein prenyltransferases, termed protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type I (GGTase-I), involved in this processing. The work has now moved into detailed mechanistic and structure-function studies on these enzymes. The focus of this project over the next 5 years will be on deciphering the molecular basis for the specificities and catalytic properties of the two enzymes using a combination of molecular biological, biochemical, and structural approaches. In addition, the cloning and molecular analysis of a recently-identified enzyme apparently involved in the metabolism of prenyl proteins will be undertaken. Elucidation of the molecular and mechanistic details of protein prenylation should provide valuable information for the design and development of inhibitors of the enzymes involved as potential chemotherapeutic agents. Additionally, the information obtained in these studies will define experimental systems that should greatly facilitate future investigations into protein prenylation and the role of this process in cellular signaling events and oncogenesis.